Validation of transformative MALDI-MS-based inhibitor screening technologies for cancer targets. Mass spectrometry (MS) has a long history as a transformative technology. Two specific examples include quantitative MS to measure the fate of drug compounds in biological systems and the development of proteomics techniques for protein identification and characterization. Recently MS-based applications have been demonstrated to be highly effective as the readout for high throughput screening (HTS) assays. The major advantage reported over the commonly used fluorescence and chemi-luminescence readout is the paucity of false positive or false negative readout. Other added benefits include a greatly reduced (>80%) reagent cost by using label-free substrates and the ability to multiplex assays such that multiple therapeutic targets can be screened for inhibitor hits with one pass through the compound repository thus saving millions of dollars in reagent and personnel costs. We have developed MALDI-MS based readout methods for measuring enzyme activity and inhibition for a variety of enzyme classes. However, the limited use of these methods with small test libraries has been insufficient to validate the overall utility of this readout for large screening campaigns. Thus the primary goal of this proposal is to systematically validate the reliability of the MALDI-MS readout compared to a traditional method of HTS using a library of 50,000 compounds. The measurements will include hit rates from the primary screen, the validation rate of the hits in secondary screening and other standard quality assessments common for HTS including signal to background, coefficient of variance (CVs) and Z'values. The target enzymes for these comparative assays will include two related kinases, PKC-zeta and PKC-iota, which appear to regulate two directly opposite effects on cancer initiation and development. Thus as a secondary aim of this proposal, the MALDI-MS readout will be assessed for its effectiveness to distinguish inhibitors from the compound repository that have selectivity for PKC-iota over PKC-zeta. We have shown that MALDI-MS readout is amenable to enzyme assays and inhibitor screening on a small scale with major advantages over existing readout methods. If, the MALDI-MS readout can be scaled to true HTS levels while maintaining all the advantages seen in the proof-of-concept studies, then this MS-based technology would likely transform the way we approach HTS, in much the same way as MS-based technologies have changed bioanalytical and proteomics applications over the past 15-20 years. Furthermore, by targeting kinases (a key class of regulatory enzymes whose dysregulation is often associated with cancer development) to validate the MALDI-MS readout approach, we can be assured that with the success of these studies, it will be clear that this technology can be readily reapplied to other cancer relevant kinases as well as expanded into other enzyme classes and disease categories. PUBLIC HEALTH RELEVANCE: Dysregulation of cellular function is a hallmark of cancer development and progression and can often be traced to the actions of one or a few cellular enzymes, thus these enzymes may act as primary targets to halt cancer development and progression. As such, rapid and accurate methods to evaluated inhibitors of these target enzymes represent an initial phase in the new drug discovery and development process. In this proposal, we investigate a new readout technology, based on mass spectrometry, to rapidly screen for inhibitors of a class of enzymes known to be involved in cancer development and progression.